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Novel Strategies for the treatment of Pseudomonas aeruginosa infections Stefanie Wagner, Roman Sommer, Stefan Hinsberger, Cenbin Lu, Rolf W. Hartmann, Martin Empting, and Alexander Titz J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01698 • Publication Date (Web): 23 Jan 2016 Downloaded from http://pubs.acs.org on January 28, 2016

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Journal of Medicinal Chemistry

Perspective for J. Med. Chem.

Novel Strategies for the Treatment of Pseudomonas aeruginosa Infections

Stefanie Wagner1,2#, Roman Sommer1,2#, Stefan Hinsberger2,3, Cenbin Lu2,3, Rolf W. Hartmann2,3, Martin Empting2,3*, Alexander Titz1,2*

1

Chemical Biology of Carbohydrates, Helmholtz Institute for Pharmaceutical Research

Saarland (HIPS), D-66123 Saarbrücken, Germany 2

Deutsches Zentrum für Infektionsforschung (DZIF), Standort Hannover-Braunschweig,

Germany 3

Drug Design and Optimization, Helmholtz Institute for Pharmaceutical Research Saarland

(HIPS), D-66123 Saarbrücken, Germany

* Corresponding authors Helmholtz Institute for Pharmaceutical Research Saarland, D-66123 Saarbrücken, email: [email protected], [email protected]

#

Both authors contributed equally.

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Abstract:

Infections with Pseudomonas aeruginosa have become a concerning threat in hospitalacquired infections and for cystic fibrosis patients. The major problem leading to high mortality lies in the appearance of drug-resistant strains. Therefore, a vast number of approaches to develop novel anti-infectives is currently pursued. These diverse strategies span from killing (new antibiotics) to disarming (anti-virulence) the pathogen. Particular emphasis lies on the development of compounds that inhibit biofilms formed in chronic infections to restore susceptibility towards antibiotics. Numerous promising results are summarized in this perspective. Antibiotics with a novel mode of action will be needed to avoid cross resistance against currently used therapeutic agents. Importantly, antivirulence drugs are expected to yield a significantly reduced rate of resistance development. Most developments are still far from the application. It can however be expected that combination therapies, also containing anti-virulence agents, will pave the way towards novel treatment options against P. aeruginosa.


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1. Introduction Pseudomonas aeruginosa, a rod-shaped Gram-negative bacterium, is a common cause of hospital-acquired infections.1 Although typically not affecting healthy individuals, it can colonize basically any part of the human body that holds sufficient humidity to provide a niche.2 In the healthcare setting, P. aeruginosa is involved in around 8-10% (51’000 reported cases in the US in 2013) of all healthcare-associated infections.3,4 Alarmingly, in about 13% of these cases multidrugresistant strains were observed and an increasing number of pan-drug-resistant specimen have been reported which cannot be treated with any of the anti-pseudomonal antibiotics available in the clinic.3 P. aeruginosa is the dominant pathogen in cystic fibrosis (CF) lungs colonizing 80% of patients by the age of 18.5 Importantly, the bacterial infection is considered the major cause of death for patients suffering from this genetic disorder.6 Other frequent sites of infection are the bloodstream, the urinary tract, surgical sites as well as burn wounds.7,8 In case of urinary tract infections (UTIs), an important predisposing factor is catheterization of the urinary tract as the instillation process may damage the mucosal layer enabling the bacteria to overcome natural biological barriers of the host.9 Importantly, the ability of P. aeruginosa to efficiently adhere to surfaces and form longlasting biofilms leads to a higher incidence of UTIs in patients with long-term bladder catheterization.10 In a similar fashion, other indwelling healthcare items like coronary stents or endotracheal tubes also bear the risk for medical-device-associated infections by P. aeruginosa.11,12 In immunocompromised patients, any of these infections by the title pathogen can be severe and result in serious complications associated with rates of high morbidity and possible mortality.3

1.1 Current Treatments and Medical Need: Antibiotics P. aeruginosa infections are usually treated with antibiotics. These antibiotics induce bacterial growth retardation or cell death, they are bacteriostatic or bactericidal, respectively. They act by inhibition of pivotal enzymes or disturbing membrane integrity.13 In general there are five main points of attack of antibiotic agents comprising the inhibition of cell wall synthesis, protein



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synthesis, and nucleic acid synthesis as well as the interference with bacterial metabolism or cell membranes.14 Several antibiotic classes acting via one of these mechanisms are in current use for the treatment of P. aeruginosa infections, especially aminoglycosides (e.g., tobramycin), fluoroquinolones (e.g., ciprofloxacin), polymyxins (e.g., colistin), and the β-lactam antibiotics cephalosporins (e.g., ceftazidime), carbapenems (e.g., meropenem), penicillins plus β-lactamase inhibitors (e.g., piperacillin-tazobactam), and the monobactam aztreonam.15,16 For every type of infection (e.g., pneumonia, bacteremia, meningitis) recommendations exist suggesting a specific agent or in most of the cases a combination of at least two antibiotics.15,16 As the clinically used antibiotics expose bacteria to an intense selective pressure, the known standard treatments are hampered by resistances emerging in a rather short time.17 For example in a recent study, Parmar et al.18 observed that about 23% of P. aeruginosa clinical isolates from a tertiary care hospital in Gujarat (India) were resistant to all 12 routine anti-pseudomonal drugs tested, such as ciprofloxacin. This growing number of resistant strains requires the analysis of patient isolates for susceptibility profiles and determination of an appropriate treatment.19,20 Mechanisms of antibiotic resistance are manifold (reviewed in Hancock et al.).4 For example, derepression of chromosomal as well as expression of plasmid-mediated β-lactamases lead to enzymatic degradation of penicillins and cephalosporins. Further resistance against these classes of antibiotics can be acquired through upregulation of the multi-drug efflux pumps MexAB-OprM and MexCD-OprJ. Efflux pump overexpression has also been reported as a resistance determinant against aminoglycosides, quinolones and carbapenems (MexXY, MexEF-OprN and others). Additionally, mutations at the drug targets themselves (e.g., GyrA/GyrB for quinolones) or changes in the lipopolysaccharide structure (LPS for polymyxins) can confer antibiotic resistance. To combat the resistant pathogens there is an urgent need of innovative antibiotics acting via novel mechanisms or targets. In this context, several new anti-pseudomonal compounds and targets have been described in the last few years, but so far only few compounds have come into clinical trials.21,22


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1.2 Virulence and Pathogenicity-determining Factors as Targets for Therapy Recently, a promising alternative to the classical antibiotic approach has been coined and is now known as anti-virulence therapy. This intervention neither leads to bacterial growth inhibition nor to bacterial cell death, but it blocks their pathogenicity.13 As a consequence, such compounds are expected to evoke a reduced rate of resistance develepment.23 Usually, a healthy human individual does not suffer from P. aeruginosa infections. It is believed that robust natural barriers provided by intact tissue, epithelial layers, the adaptive and innate immune system, as well as the plethora of naturally occurring and competing microbes give a sufficient protection against this ubiquitous bacterium. However, in situations when defense mechanisms (burn wound) or mucociliary clearance (cystic fibrosis) is impaired, P. aeruginosa may overwhelm the host.24,25 To this end, it is equipped with an arsenal of pathogenicity determinants often referred to as virulence factors.26 These are diverse in structure, function, localization as well as regulation and can be roughly divided into those virulence factors needed for acute and those employed in the chronic state of infection. To establish an infection in a host, it has to be invaded or colonized. Therefore, P. aeruginosa first locally adheres to sites where the host defense lines are already disrupted (e.g., via trauma, burn, surgery, other causes of inflammation, etc.). This event is mediated mainly by cell-associated virulence factors like type IV pili or the carbohydrate-binding proteins (lectins).26 In an acute infection scenario, an increased production of extracellular virulence factors further damages host tissue which ultimately enables blood vessel invasion and dissemination leading to a life-threatening systemic spread of the bacteria. Secreted virulence factors, like elastases, alkaline proteases, phospholipases, rhamnolipids, leukocidin, hydrogen cyanide, cytotoxins, pyocyanin, or siderophores concertedly facilitate this process which is mainly regulated through bacterial quorum sensing (QS).26 Notably, the signal molecules cyclic di-GMP, cyclic AMP or small RNAs play an important role in the regulation of bacterial virulence and all these regulatory pathways are highly interconnected.27 In a next step, the adhered colonies form biofilms providing an effective protection against the host immune response. A biofilm consists of bacteria embedded in an extracellular matrix which contains rhamnolipids, extracellular DNA, polysaccharides and proteins, i.e., a biofilm can be 5 ACS Paragon Plus Environment


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considered as a hydrated biopolymeric framework containing a cocktail of virulence factors.28 Upon establishing a chronic infection P. aeruginosa usually undergoes a genetic adaptation resulting in a phenotype with pronounced mucoidity.29,30 Among many other factors, this also involves an increased expression of the virulence factor alginate leading to increased resistance towards host defense mechanisms as well as antibiotic treatment.29,31,32 Finally, further maturation of the biofilm is accompanied by a lowered production of extracellular virulence factors needed for acute infection.26 However, a persistent P. aeruginosa infection may cause a chronic local inflammation with deleterious effects for the surrounding host tissue.33 Continuous long-term damage of chronically infected airways is thus the major cause of death in cystic fibrosis patients.6 Virulence mechanisms are essential for host infection and persistence, and therefore selectively targeting these bacterial traits has emerged as an attractive strategy in drug design.13 In this regard, ‘anti-virulence’ agents or ‘pathoblockers’ may directly interfere with the biosynthesis, secretion, or function of specific virulence factors or disturb/disrupt higher regulatory systems which control the production of virulence arsenals.34

1.2.1 Secretion Systems as Targets for Therapy Gram-negative bacteria possess protein secretion systems, which are molecular nanomachines spanning the two bacterial membranes to release proteins and enzymes among which are numerous virulence factors (Figure 1). P. aeruginosa harbors five of the so far six known secretion systems in Gram-negative bacteria, termed Type I to Type VI secretion systems, with absence of the Type IV secretion system.35 These can be classified into two-step and one-step secretion systems. The latter are nanomachines spanning the two bacterial membranes, whereas for the two-step secretion proteins transit through the periplasmic space before they are passing the outer membrane. Secreted proteins are often involved in the hydrolysis of complex structures into digestable nutrients and also supply the cell with essential inorganic ions from the environment. Protein secretion systems play an important role in bacterial virulence. Pathogens use these nanomachines to either release virulence factors into their environment (e.g., elastases LasA and LasB), or direct translocation into the host cell cytosol.36,37 Hence, secretion systems themselves have become validated anti-virulence targets. 6 ACS Paragon Plus Environment

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Figure 1: Schematic representation of the secretion systems present in P. aeruginosa. PM plasma membrane, OM outer membrane, IM inner membrane.

35

1.2.2 Cell Adhesion and Biofilm Formation as Target for Therapy The resistance of P. aeruginosa against antibiotics also results from its potential for biofilm formation, in addition to other resistance mechanisms described above. In a biofilm, bacteria are protected against host immune defense and antibiotic treatment. The biofilm development of P. aeruginosa is proposed as a five stage process (Figure 2) based on in vitro observations.38,39 The first step is the reversible attachment of single bacterium to a surface. After a critical local density of bacteria is achieved (irreversible attachment), P. aeruginosa starts to produce an extracellular polymeric substances (EPS) containing matrix (early maturation) which encloses the final mushroom shaped mature biofilm (late maturation). To colonize further regions, the bacteria are then able to disperse from the biofilm in the last stage (dispersion). Notably, biofilms found in vivo are significantly smaller in size and mushroom-shaped structures have not been observed.40



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Figure 2: The five stages of biofilm formation: (I) reversible attachment, (II) irreversible attachment, (III) early maturation, (IV) late maturation and (V) dispersion.

41

Bacteria embedded in a biofilm are surrounded by a self produced EPS matrix, which is a dynamic complex mixture consisting of rhamnolipids, exopolysaccharides, extracellular DNA (eDNA) and several proteins.42 P. aeruginosa produces three exopolysaccharides, Pel, Psl and Alginate.43 These polysaccharides effect the stability and architecture of the biofilm and have a high impact on adherence, cell-cell interaction mechanisms and virulence of the bacteria.42,44 The production and release of extracellular non-enzymatic proteins contributes considerably to biofilm formation of P. aeruginosa and leads to stability of the biofilm.42 Several adhesins such as flagellar FliD,45 type IV pili,46 CdrA,47 and the lectins LecA48,49 and LecB50,51 play an important role in host recognition, contribute to initial attachment and stabilize the EPS matrix by interfering with the polysaccharides. The resulting sticky EPS hydrogel can act as a physical barrier by lowering the diffusion of some antibiotics and host immune defense mechanisms.42 Furthermore, the same EPS matrix lowers the concentration of nutrients (e.g., oxygen or carbon source) within the biofilms.52 Due to this nutritional gradient bacteria in deeper layers of the biofilm reduce their metabolic activity. Antibiotics, e.g., ciprofloxacin, gentamycin or meropenem, which interfere with metabolic processes as replication, translation or cell wall synthesis, are therefore less active towards bacteria in a biofilm.53,54 In contrast, it has been reported that metabolically active P. aeruginosa in the upper layer of a biofilm can adapt to some antibiotics interfering with the membrane like colistin.55 Whereas this subpopulation is not affected, colistin can kill dormant bacteria in a biofilm.55 However, a combined antimicrobial treatment is not able to eradicate all bacteria and the


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so-called "persisters" remain. Whether these cells have a different phenotype or are the most resistant cells within one population is still under discussion.53

Since the classical antibiotics are not sufficient in treatment of bacterial infections new paradigms for antimicrobial therapy are pursued.13 To prevent the appearance of MDR by adaptation, bacterial targets of non-essential processes are of interest. Here, QS system of P. aeruginosa, biogenesis of adhesive organelles (e.g., pilicides) or the carbohydrate adhesins are promising targets against bacterial biofilm formation (reviewed in56).

1.3 Regulation of Virulence as Target for Therapy 1.3.1 Quorum Sensing Quorum sensing (QS) is a cell-to-cell communication network that enables bacteria to take cell density-dependent collective decisions thereby synchronizing the whole bacterial community to act as a multicellular organism. A typical QS system is composed of a signal molecule termed autoinducer (AI), a synthase producing the AI, and a receptor/transcriptional regulator detecting the signal. The binding of this signal molecule by its cognate receptor activates the transcription of several genes including those for AI biosynthesis, enabling a positive autoinducing loop. Generally, at a low cell density the AI reach only low concentrations and the receptor is activated at a basal level. As the local population increases, the AI reaches a threshold concentration leading to a full activation of the receptor and up-regulation of target genes. Remarkably, the QS-regulated genes are normally not involved in bacterial growth, but closely linked to pathogenicity, including the production of virulence factors, the formation of biofilm, swarming, swimming and twitching motility.57 P. aeruginosa possesses three main QS circuits denoted as las,58 rhl59,60 and pqs61 (Figure 3). Both las and rhl utilize acylated homoserine lactones (AHLs) for signaling. The synthase of las QS termed LasI produces AI N-3-oxododecanoyl-L-homoserine lactone (3-oxo-C12-HSL), which stimulates the receptor LasR. In an analogous fashion, the enzyme RhlI synthesizes N-butanoyl-Lhomoserine lactone (C4-HSL), which activates the rhl receptor RhlR. For the pqs QS, the synthases PqsABCDEH produce the Pseudomonas Quinolone Signal (PQS) and its precursor 29 ACS Paragon Plus Environment


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heptyl-4-hydroxyquinoline (HHQ), two prominent members of the 2-alkyl-4-hydroxyquinoline (HAQ) family,62 as AIs that can be detected by their cognate receptor MvfR/PqsR.63,64 Notably, all the networks are intertwined and depend on each other (Figure 3).65,66,66-68 Recently, a fourth QS pathway signaled by IQS was disclosed.69

Figure 3: Pathways involved in the regulation of acute toxicity and biofilm lifestyle of P. aeruginosa. Arrows indicate induction, blunt bars indicate downregulation.

Since QS controls a large set of pathogenicity-associated genes of P. aeruginosa, interception of the cell-to-cell communication by QS inhibitors (QSIs) is a promising approach for an anti-virulence therapy to disarm rather than kill the pathogens. Generally, three strategies can be applied to interrupt QS signaling: 1) blockade of AI production via inhibiting AI synthases e.g., LasI, RhlI, or


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PqsA-D, 2) inactivation of signal transmission via degradation or sequestering of AIs, and 3) interference with signal receptors e.g., LasR, RhlR and MvfR/PqsR.70

1.3.2 Cyclic-di-GMP signalling Cyclic diguanylate monophosphate (c-di-GMP) is a second messenger in bacteria that controls the switching between motile (planktonic) and sessile (biofilm) lifestyles, virulence of pathogens and other cellular functions (for reviews see71-73). The genes involved in its signaling pathways are conserved in bacteria but absent in animal species, rendering it as possible anti-infective target.74 High concentrations of c-di-GMP induce the synthesis of adhesins and exopolysaccharides (Pel, Psl, alginate) in P. aeruginosa and at the same time inhibit various forms of motility (Figure 3). In addition, c-di-GMP is involved in virulence control of pathogenic bacteria.75 C-di-GMP is synthesized by diguanylate cyclases (DGC), which harbor the specific amino acid sequence GGDEF in their active sites, and hydrolyzed by phosphodiesterases (PDE, containing an EAL or HD-GYP amino acid sequence domain) into linearized pGpG which can be further degraded to GMP. In P. aeruginosa many proteins carrying the GGDEF, EAL or even both domains are expressed,76 indicating that various input signals exist that control c-di-GMP signaling. To date, not all pathways are fully understood. Despite the high number of DGC and PDE enzymes, so far, only four effector proteins are characterized, i.e., proteins that bind c-di-GMP and activate or inhibit expression of the target genes or alter their function in response to c-di-GMP binding. While QS is important for the formation of a fully mature biofilm, c-di-GMP was shown responsible for the initial adherence and early stages of biofilm formation, as a switch from a planktonic to a biofilm lifestyle. Christensen et al.77 showed that decreasing the c-di-GMP level in P. aeruginosa by over-expressing a PDE enzyme resulted in biofilm dispersal, validating c-di-GMP signaling as a target for new anti-infectives.

The sections above summarize essential traits of P. aeruginosa important for its pathogenicity. In addition to the development of novel antibiotic agents with bacteriostatic and bactericidal effects 11 ACS Paragon Plus Environment


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(see Section 2.1), the mentioned virulence mechanisms provide novel targets to interfere with P. aeruginosa infectivity. Section 2.2 reports recent advances in this field. Finally, we highlight promising drug combinations that are currently in development (Section 2.3), and the importance of appropriate model test systems in drug research against Pseudomonas infections (Section 2.4).

2. Novel Therapies 2.1 Novel Antibiotics To overcome the general resistance problem, in the last few years several new antibiotics were developed and approved, also comprising drugs active against P. aeruginosa.21 Especially in the classes of quinolones, cephalosporins and carbapenems novel agents were found. Furthermore, new β-lactamase inhibitors are underway and raise hopes that the activity of known β-lactams can be restored against β-lactamase-producing strains.78 However, most of these “new” antibiotics do not exhibit a novel mode of action or target a novel binding site, but belong to well-known classes. However, beside these established groups of therapeutics other innovative antibiotic strategies and compounds have been developed.

2.1.1 The Argyrins - targeting elongation factor G The argyrins 1-8 are a class of 8 naturally occurring cyclic peptides isolated by Sasse, Höfle and co-workers from the myxobacterium Archangium gephyra (Figure 4).79,80 Numerous properties have been described and besides cytotoxic activity presumably via proteasome inhibition and immunomodulatory

effects,

these

compounds

show

good

antibiotic

effects

against

P.

aeruginosa.81-83 A structure-activity-relationship for the natural argyrins A-H shows IC50 values in the sub-microgram per milliliter range with argyrin B as most potent derivative (2, IC50 0.08 µg/mL). Already in 1996, two cyclic peptides with a similar sequence but a regional isomer of the methoxy substituent at the tryptophan were reported as antibiotics A21459A and B by the Marion Merell Dow Research Institute.81,84 Later, it was shown that antibiotics A21459A and B are in fact identical to argyrin A and B, respectively, with a revised position of the methoxy substituent.80 The first total synthesis for an argyrin was reported by Ley85 and co-workers for argyrin B in 18 linear steps, 12 ACS Paragon Plus Environment

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followed by an alternative approach for argyrin F (6) by the Kalesse86 group. The latter work also yielded a number of non-natural derivatives of argyrin F which were tested for proteasomal activity. In 2011, Wu et al. disclosed their strategy for the total synthesis of argyrins A and E with a reduced length in the number of linear steps to 14.87 Chen et al. recently synthesized a number of derivatives of argyrin A with modified substituents at the 4’-methoxy-tryptophan residue.88 Substitution of the 4’-methoxy group by halogens or other substituents in various positions abolished its antibacterial activity, whereas the 5’-methoxy-tryptophan derivative displayed only slightly reduced activity against P. aeruginosa PAO1. Bielecki et al. showed a high efficacy of argyrin A against a panel of multi-drug resistant (MDR) strains of P. aeruginosa.89 Recently, it was shown by two independent groups, that the argyrins bind to elongation factor G (EF-G) as conserved target in bacteria (P. aeruginosa and other) and eukaryotes.89,90 The co-crystal structure of argyrin B (2) and P. aeruginosa EF-G1 encoded by its gene fusA1 now provides structural information at atomic resolution for further optimization.90 Due to its dual activity against the human proteasome and bacterial targets, selectivity of this class of compounds should be considered in the development of future antibacterials.



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Figure 4: Naturally occurring argyrins with potent anti-pseudomonal activity.

2.1.2 LpxC inhibitors The protein UDP-3-O-(acyl)-N-acetylglucosamine deacetylase (LpxC) is a cytosolic zincdependent enzyme that catalyzes a rate-determining, irreversible step in the biosynthesis of lipid A.91,92 As lipid A is an essential element of the outer membrane of most Gram-negative bacteria, including P. aeruginosa, LpxC represents an attractive target for novel antibiotic agents.93 Several LpxC inhibitors have been described in the last decades.94-97 A well-documented prototype is CHIR-090 (9, Figure 5).96,98,99 Many further LpxC inhibitor classes based on or at least possessing a certain structural similarity to 9 have been developed subsequently.100-106 9 and its analogues consist of a zinc-binding hydroxamate group and a hydrophobic tail, two important structural features present in most of the LpxC inhibitors, while the core linking the two key features varies between the different classes. Most of the described inhibitors, especially 9, have been extensively characterized concerning their binding modes, IC50 and MIC values for different bacterial species, 14 ACS Paragon Plus Environment

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including numerous P. aeruginosa strains. For example, Brown et al.100 reported the methylsulfone-containing hydroxamic acid 10 inhibiting the P. aeruginosa LpxC enzyme with an IC50 of 1 nM and with a MIC90 of 2 µg/mL for 91 tested P. aeruginosa strains. In vivo evaluation in rats revealed a rather high clearance (58 mL/min/kg) following i.v. dosing. Additionally, 10 exhibits significant binding to human plasma protein. Despite these drawbacks, in a murine acute systemic infection model the compound was able to reduce the P. aeruginosa burden in the spleen (ED50 35 mg/kg) and to increase the survival at 96 h after the infection with the pathogen (PD50 63 mg/kg).100

Tomaras and colleagues107 provided a detailed characterization of methylsulfone-containing inhibitor LpxC-4 (11)103 and could demonstrate that resistance against 11 occurs at frequencies comparable to those of approved antibiotic drugs. 11 was further tested in an acute (48 h) mouse pneumonia model with twice-daily dosing, revealing efficacy against P. aeruginosa PA-1950 with an ED50 of

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